Jin-Woo Bae

Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.

Although commensalism with gut microbiota exists in all metazoans, the host factors that maintain this homeostatic relationship remain largely unknown. We show that the intestinal homeobox gene Caudal regulates the commensal-gut mutualism by repressing nuclear factor kappa B–dependent antimicrobial peptide genes. Inhibition of Caudal expression in flies via RNA interference led to overexpression of antimicrobial peptides, which in turn altered the commensal population within the intestine. In particular, the dominance of one gut microbe, Gluconobacter sp. strain EW707, eventually led to gut cell apoptosis and host mortality. However, restoration of a healthy microbiota community and normal host survival in the Caudal-RNAi flies was achieved by reintroduction of the Caudal gene. These results reveal that a specific genetic deficiency within a host can profoundly influence the gut commensal microbial community and host physiology.

An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice

Background Recent evidence indicates that the composition of the gut microbiota contributes to the development of metabolic disorders by affecting the physiology and metabolism of the host. Metformin is one of the most widely prescribed type 2 diabetes (T2D) therapeutic agents. Objective To determine whether the antidiabetic effect of metformin is related to alterations of intestinal microbial composition. Design C57BL/6 mice, fed either a normal-chow diet or a high-fat diet (HFD), were treated with metformin for 6 weeks. The effect of metformin on the composition of the gut microbiota was assessed by analysing 16S rRNA gene sequences with 454 pyrosequencing. Adipose tissue inflammation was examined by flow cytometric analysis of the immune cells present in visceral adipose tissue (VAT). Results Metformin treatment significantly improved the glycaemic profile of HFD-fed mice. HFD-fed mice treated with metformin showed a higher abundance of the mucin-degrading bacterium Akkermansia than HFD-fed control mice. In addition, the number of mucin-producing goblet cells was significantly increased by metformin treatment (p<0.0001). Oral administration of Akkermansia muciniphila to HFD-fed mice without metformin significantly enhanced glucose tolerance and attenuated adipose tissue inflammation by inducing Foxp3 regulatory T cells (Tregs) in the VAT. Conclusions Modulation of the gut microbiota (by an increase in the Akkermansia spp. population) may contribute to the antidiabetic effects of metformin, thereby providing a new mechanism for the therapeutic effect of metformin in patients with T2D. This suggests that pharmacological manipulation of the gut microbiota in favour of Akkermansia may be a potential treatment for T2D.

Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food

ABSTRACT Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing

Little is known about the archaeal diversity of fermented seafood; most of the earlier studies of fermented food have focused on lactic acid bacteria (LAB) in the fermentation process. In this study, the archaeal and bacterial diversity in seven kinds of fermented seafood were culture-independently examined using barcoded pyrosequencing and PCR–denaturing gradient gel electrophoresis (DGGE) methods. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers, using different primers for Archaea or Bacteria. Because PCR–DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results were compared with the results of the barcoded pyrosequencing analysis. A total of 13 372 sequences were retrieved from 15 898 pyrosequencing reads and were analyzed to evaluate the diversity of the archaeal and bacterial populations in seafood. The most predominant types of archaea and bacteria identified in the samples included extremely halophilic archaea related to the family Halobacteriaceae; various uncultured mesophilic Crenarchaeota, including Crenarchaeota Group I.1 (CG I.1a and CG I.1b), Marine Benthic Group B (MBG-B), and Miscellaneous Crenarchaeotic Group (MCG); and LAB affiliated with genus Lactobacillus and Weissella. Interestingly, numerous uncultured mesophilic Crenarchaeota groups were as ubiquitous in the fermented seafood as in terrestrial and aquatic niches; the existence of these Crenarchaeota groups has not been reported in any fermented food. These results indicate that the archaeal populations in the fermented seafood analyzed are diverse and include the halophilic and mesophilic groups, and that barcoded pyrosequencing is a promising and cost-effective method for analyzing microbial diversity compared with conventional approaches.

Proteobacteria: microbial signature of dysbiosis in gut microbiota

Recent advances in sequencing techniques, applied to the study of microbial communities, have provided compelling evidence that the mammalian intestinal tract harbors a complex microbial community whose composition is a critical determinant of host health in the context of metabolism and inflammation. Given that an imbalanced gut microbiota often arises from a sustained increase in abundance of the phylum Proteobacteria, the natural human gut flora normally contains only a minor proportion of this phylum. Here, we review studies that explored the association between an abnormal expansion of Proteobacteria and a compromised ability to maintain a balanced gut microbial community. We also propose that an increased prevalence of Proteobacteria is a potential diagnostic signature of dysbiosis and risk of disease.

Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses

ABSTRACT Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

Comparative Analysis of Korean Human Gut Microbiota by Barcoded Pyrosequencing

Human gut microbiota plays important roles in harvesting energy from the diet, stimulating the proliferation of the intestinal epithelium, developing the immune system, and regulating fat storage in the host. Characterization of gut microbiota, however, has been limited to western people and is not sufficiently extensive to fully describe microbial communities. In this study, we investigated the overall composition of the gut microbiota and its host specificity and temporal stability in 20 Koreans using 454-pyrosequencing with barcoded primers targeting the V1 to V3 region of the bacterial 16S rRNA gene. A total of 303,402 high quality reads covered each sample and 8,427 reads were analyzed on average. The results were compared with those of individuals from the USA, China and Japan. In general, microbial communities were dominated by five previously identified phyla: Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Proteobacteria. UPGMA cluster analysis showed that the species composition of gut microbiota was host-specific and stable over the duration of the test period, but the relative abundance of each member fluctuated. 43 core Korean gut microbiota were identified by comparison of sequences from each individual, of which 15 species level phylotypes were related to previously-reported butyrate-producing bacteria. UniFrac analysis revealed that human gut microbiota differed between countries: Korea, USA, Japan and China, but tended to vary less between individual Koreans, suggesting that gut microbial composition is related to internal and external characteristics of each country member such as host genetics and diet styles.

Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host.

ABSTRACT Insects are the most abundant animals on Earth, and the microbiota within their guts play important roles by engaging in beneficial and pathological interactions with these hosts. In this study, we comprehensively characterized insect-associated gut bacteria of 305 individuals belonging to 218 species in 21 taxonomic orders, using 454 pyrosequencing of 16S rRNA genes. In total, 174,374 sequence reads were obtained, identifying 9,301 bacterial operational taxonomic units (OTUs) at the 3% distance level from all samples, with an average of 84.3 (±97.7) OTUs per sample. The insect gut microbiota were dominated by Proteobacteria (62.1% of the total reads, including 14.1% Wolbachia sequences) and Firmicutes (20.7%). Significant differences were found in the relative abundances of anaerobes in insects and were classified according to the criteria of host environmental habitat, diet, developmental stage, and phylogeny. Gut bacterial diversity was significantly higher in omnivorous insects than in stenophagous (carnivorous and herbivorous) insects. This insect-order-spanning investigation of the gut microbiota provides insights into the relationships between insects and their gut bacterial communities.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

Comparing microarrays and next-generation sequencing technologies for microbial ecology research

Recent advances in molecular biology have resulted in the application of DNA microarrays and next-generation sequencing (NGS) technologies to the field of microbial ecology. This review aims to examine the strengths and weaknesses of each of the methodologies, including depth and ease of analysis, throughput and cost-effectiveness. It also intends to highlight the optimal application of each of the individual technologies toward the study of a particular environment and identify potential synergies between the two main technologies, whereby both sample number and coverage can be maximized. We suggest that the efficient use of microarray and NGS technologies will allow researchers to advance the field of microbial ecology, and importantly, improve our understanding of the role of microorganisms in their various environments.